Biliary tract cancer (BTC) has proven challenging to treat and manage due to its poor sensitivity to conventional therapies and the inability to prevent or detect early tumor formation. All of these factors render gallbladder cancer nearly incurable with a five year survival rate of only 5-21%. An estimated 20,000 new cases of liver and biliary tract cancer are diagnosed annually in the United States and each year 4,000 patients will die of BTC. BTC is the 2nd most common primary hepatobiliary cancer, after hepatocellular cancer, with approximately 8,000 cases reported annually. Nearly 2/3rds of these arise in the gallbladder, making it the most common BTC and 5th most common gastrointestinal tract cancer, while the remainder (cholangiocarcinoma) originate in the bile ducts and periampullary region. To date, very few studies have attempted to decipher the molecular and cellular mechanism(s) involved in the development of gallbladder carcinoma, and very little is known regarding the sequence of events leading to the development of BTC. A limiting factor is the lack of an animal model for the spontaneous development of BTC. Presently available animal models are based on exposure to chemical carcinogens. In these models, the latency between the treatment and tumor development is long and the tumor incidence is relatively low. We recently developed the BK5.erbB2 transgenic mice where expression of a rat erbB2 cDNA is targeted to the basal layer of multiple epithelial tissues, including the biliary tract epithelium, by a bovine keratin 5 (BK5) promoter ((1,2) and see Appended manuscripts 1 and 2). Tumors arise at various sites of the biliary tract as a consequence of elevated erbB2 expression. In particular, papillary adenocarcinoma of the gallbladder develops in 90% of the homozygous BK5.erbB2 transgenic mice by 3-4 months of age. The BK5.erbB2 transgenic mouse line represents the first genetically engineered mouse model for BTC. We have shown that the BK5.erbB2 transgenic line is a valid model for investigating the mechanism(s) of development of gallbladder carcinoma and other BTCs ((2) and manuscript in preparation). We have found that protein levels of erbB2 as well as protein levels of epidermal growth factor receptor (EGFR) are elevated in the gallbladder in BK5.erbB2 transgenic mice. Both erbB2 and EGFR are constitutively activated in these tumors. In addition, we have found that cyclooxygenase-2 (COX-2) (mRNA and protein) levels are also up-regulated. Upregulation of erbB2, EGFR and COX-2, which are often associated with many human cancers including BTC, are observed in the gallbladder carcinoma that develop in the BK5.erbB2 mice. In this proposal we will elucidate the mechanism(s) of development of gallbladder carcinoma including cholangiocarcinoma. Our hypothesis is that upregulation of COX-2 is crucial to the development of BTC in the BK5.erbB2 transgenic mice and that this involves the up-regulation of specific genes, particularly Muc4, downstream of COX-2. The results provided in this proposed study would present critical clues for novel therapeutic or chemopreventive strategies using new or conventional drugs that selectively target the specific augmented molecule(s) in BTC. The Specific Aims are: 1. Establish the requirement for COX-2 overexpression in the development of BTC; 2. Determine the critical involvement of Akt and MAPK in the upregulation of COX-2 in gallbladder carcinoma that develop in BK5.erbB2 mice, and 3. Establish the involvement of Muc4 as a critical downstream target of COX-2 in the development of BTC in BK5.erbB2 mic.